The present invention relates generally to a method and composition for separating subpopulations of cells from heterogeneous cell suspensions. More particularly, the present invention relates to separation and isolation of lymphocytes and accessory cells.
The suspended particles of blood comprise approximately 45 percent of the total volume of blood. The suspended particles include red blood cells (erythrocytes), white blood cells (leukocytes) and platelets (thrombocytes). Although the great majority of the cells in blood are red blood cells, the white blood cells are extremely important since they are the body's primary defense against infections.
There are basically three types of white blood cells: the lymphocytes, the granulocytes and the monocytes. The lymphocytes are further classified as T lymphocytes, B lymphocytes and Null lymphocytes. The T-lymphocytes may be further classified into subpopulations identified a helper T-lymphocytes and suppressor T-lymphocytes. The granulocytes are also further classified as neutrophils, eosinophils and basophils. The white blood cells are also classified generally as lymphocytes and accessory cells. The lymphocytes include the T, B ad Null lymphocytes. The granulocytes and monocytes are classified as accessory cells.
Past progress in understanding the cellular immunology of the white blood cells and future progress in further understanding of the white blood cells is dependent upon our ability to separate and isolate the various lymphocytes and accessory cells to allow their identification and analysis. Due to the importance of separating and distinguishing white blood cells subpopulations, a wide variety of cell separation procedures have been developed. Common techniques presently employed for separating and isolating white blood cell populations include adherence type processes in which one or more of the white blood cell types are removed by adherence to various materials such as Sephadex and nylon wool. Other procedures have utilized density gradients to separate the white blood cell populations into their respective classes by size and density differences.
In order for a cell separation method to be useful, the separation technique must be simple and produce high, representative yields of the desired cells with little or no contamination by other white blood cell types. The separation technique should also be easily reproducible and not adversely affect the behavior of the recovered white cell population. Although the above mentioned cell separation methods are fairly successful, these methods still require a considerable number of steps which are not only time consuming but increase the chance for sample mix-up.
Recently, a new cell isolation and separation method has been developed which is based upon the specific cell surface markers present in each of the white blood cell types. Basically, the method involves treating the heterogenous population of white blood cells (which generally will also include some red blood cells) with one or more specific antibodies which recognize and become attached to one or more of the white cell subpopulations. Complement is then added to the cells to lyse the antibody tagged cells. The lysed cells are then separated from the remaining non-lysed heterogeneous white cell population. This negative selection type method has shown some promise; however, disadvantages have included suitable sources of antisera and complement which provide the desired lysing of the white cell subpopulation that is to be removed from the heterogeneous white cell population. Also, separate steps involving antibody treatment, complement lysing of cells and separation of the lysed cells from the desired cell subpopulation are required.
In view of the importance of optimum separation and isolation of the various white cell elements, it is desirable to provide isolation methods and reagents useful in such methods which simplify cell isolation procedures, enhance cell separation and provide optimum yields of desired viable cells.